LAG3 (CD233) is an inhibitory receptor that plays a critical role in controlling T cell tolerance, preventing autoimmunity and limiting immune-mediated tissue damage. It is highly upregulated on exhausted T cells in tumors and in chronic viral infections, limiting the development of sterilizing immunity. Consequently, LAG3 is now a major immunotherapeutic target for the treatment of cancer and other diseases, with multiple clinical trials ongoing. Despite extensive analysis of LAG3 for over 30 years and multiple LAG3 targeting therapeutics in the clinic, very little is known about the mechanism of action of LAG3 and thus how to develop optimal LAG3- targeting therapeutics, especially agonists. While it is assumed that the primary and perhaps only functionally relevant ligand for LAG3 is MHC class II, there are several observations including our recent preliminary studies that suggest that there are additional biophysical requirements for LAG3 to function. AIM 1: Define and compare LAG3 interaction with its ligands. We will address 3 questions: (A) Which domains and residues mediate LAG3 interaction with its ligands? (B) Can the association of LAG3 with its ligands be disrupted by different anti-LAG3 Abs? (C) What LAG3 residues mediate ligand interaction? AIM 2: Determine crystal structures of LAG3 and of LAG3:Ab and LAG3:MHC class II complexes. We will address the structural basis for the biological function of LAG3 by asking 3 questions: (A) What unique structural features of LAG3 distinguish it from CD4? (B) What is the structure of LAG3:Ab complexes? (C) What is the structure of LAG3:MHC class II complexes? AIM 3: Determine the functional impact of LAG3 interaction with its ligands. We will address 3 questions: (A) Is LAG3 association with its various ligands required for its function? (B) Does LAG3 interaction with its various ligands have a differential impact on its function in tumor models? (C) Is blocking the association of LAG3 with its different ligands have differential therapeutic benefit? This project will have a significant impact on our understanding of the mechanism of action of LAG3, as well as provide further insight into the generation of enhanced therapeutic reagents that block or enhance LAG3 function. Given that we have determined the function of LAG3 on regulatory and effector T cell function, have been studying LAG3 function of ~19 years, have available to us a variety of unique tools to probe its function and have assembled a strong and technically diverse collaborative team, we are in the best position to conduct this research.